Incoherent transmission electron microscopy

ABSTRACT

A transmission electron microscope includes an electron beam source to generate an electron beam. Beam optics are provided to converge the electron beam. A specimen holder is provided to hold a specimen in the path of the electron beam. A detector is used to detect the electron beam transmitted through the specimen. The transmission electron microscope may be adapted to generate two or more images that are substantially incoherently related to one another, store the images, and combine amplitude signals at corresponding pixels of the respective images to improve a signal-to-noise ratio. Alternatively or in addition, the transmission electron microscope may be adapted to operate the specimen holder to move the specimen in relation to the beam optics during exposure or between exposures to operate the transmission electron microscope in an incoherent mode.

CLAIM FOR PRIORITY

This application is a continuation of application Ser. No. 13/759,029,filed Feb. 4, 2013, which is a continuation of U.S. Pat. No. 8,389,937,filed Jun. 7, 2011, which claims priority under 35 U.S.C. §119(e) toProvisional Application No. 61/352,243, filed Jun. 7, 2010, and is acontinuation-in-part of U.S. patent application Ser. No. 13/024,961,filed Feb. 10, 2011, which claims priority under 35 U.S.C. §119(e) toProvisional Application No. 61/303,260, filed Feb. 10, 2010, andProvisional Application No. 61/352,243, filed Jun. 7, 2010, all of whichare incorporated herein by reference in their entireties.

TECHNICAL FIELD

This application relates to incoherent electron microscopy.

BACKGROUND

There are applications in which it is desirable to use electronmicroscopy to resolve a single point-like object in a specimen. Thesingle point-like object may be, for example, a single atom or a clusterof atoms on an amorphous substrate. Electron microscopy couldtheoretically be used to sequence bases of a nucleic acid, for example,such as the bases of a strand of deoxyribonucleic acid (DNA).

Scanning transmission electron microscopy (STEM), which raster scans anelectron beam across a specimen, can be used to resolve singlepoint-like objects in an image. However, STEM typically suffers from aslow scanning time, which causes poor throughput. For example, STEM mayinvolve scanning for a time on the order of 1 μs to 10 μs per pixel ofthe image. This scanning time may be inadequate where sequentialresolution of numerous single point-like objects is desired. STEMthroughput may be inadequate, for example, for sequencing a full humangenome in a practical amount of time.

Transmission electron microscopy (TEM), unlike STEM, images the specimenin parallel. But TEM imaging can be problematic when trying to resolvesingle point-like objects because the phase-contrast information istypically not directly interpretable for this purpose. For example, alight area in a TEM image could represent either an atom or the absenceof an atom. Accordingly, although TEM may have good throughput, it doesnot typically yield the desired information about the specimen.

Thus, it is desirable to have electron microscopy that can reliablyresolve point-like objects. It is further desirable for such electronmicroscopy to have substantially high throughput. Moreover, it isdesirable for such electron microscopy to be provided at an affordablecost.

SUMMARY

In one embodiment, a transmission electron microscope comprises anelectron beam source to generate an electron beam. Beam optics areprovided to converge the electron beam. The microscope further comprisesa specimen holder to hold a specimen in the path of the electron beam. Adetector is provided to detect the electron beam transmitted through thespecimen. The transmission electron microscope of this embodiment isadapted to generate two or more images that are substantiallyincoherently related to one another, store the images, and combineamplitude signals at corresponding pixels of the respective images toimprove a signal-to-noise ratio.

In another embodiment, a method of generating an image of a specimencomprises generating an electron beam and converging the electron beam.A specimen is held in the path of the electron beam. The electron beamtransmitted through the specimen is detected to generate two or moreimages that are substantially incoherently related to one another, thetwo or more images are stored, and amplitude signals at correspondingpixels of the respective images are combined to improve asignal-to-noise ratio.

In yet another embodiment, a transmission electron microscope comprisesan electron beam source to generate an electron beam. Beam optics areprovided to converge the electron beam. The microscope further comprisesa specimen holder to hold a specimen in the path of the electron beam. Adetector is provided to detect the electron beam transmitted through thespecimen. The transmission electron microscope of this embodiment isadapted to operate the specimen holder to move the specimen in relationto the beam optics during exposure or between exposures to operate thetransmission electron microscope in an incoherent mode.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and aspectsof the transmission electron microscopes described herein and, togetherwith the description, serve to explain the principles of the invention.

FIG. 1 is a schematic diagram of an exemplary embodiment of anaberration-correcting ADF-TEM column.

FIG. 2 is a schematic diagram of an exemplary embodiment of animplementation of an incoherent illumination mode using an incoherentelectron source.

FIGS. 3 a and 3 b are schematic diagrams of exemplary embodiments ofelectron beam trajectories through a set of condenser lenses, showingdifferent amounts of demagnification.

FIG. 4 is a schematic diagram of an exemplary embodiment of a referenceversion of a TEM.

FIG. 5 is a schematic diagram of an exemplary embodiment of a dark-fieldversion of the TEM illustrated in FIG. 4.

FIG. 6 is a schematic diagram of an exemplary embodiment of animplementation in which image constituents from a tilted and scannedelectron beam are summed.

FIG. 7 illustrates an exemplary embodiment of a variation of the TEMillustrated in FIG. 6 with an annular aperture.

FIG. 8 illustrates an exemplary embodiment of a dark-field variation ofthe tilted-beam version of FIG. 6.

FIG. 9 is a set of plots illustrating a generalized version of themanner in which amplitude contrast is summed while phase contrast isdecreased to improve the image of an object being identified in thespecimen.

FIGS. 10 a and 10 b are images and histograms of two examples ofamplitude signals received by a transmission electron microscope along apreselected direction.

FIGS. 11, 12, 13, 14, 15, 16, and 17 are schematic diagrams of variousexemplary embodiments of implementation of incoherent superposition.

DETAILED DESCRIPTION

A transmission electron microscope (TEM) is able to image a specimen inparallel, thereby theoretically offering rapid and efficient throughput.As explained above, however, TEM imaging can be problematic when tryingto resolve single point-like objects because the phase-contrastinformation in the image is typically not directly interpretable forthis purpose. This problem may arise, for example, when trying to imagesingle atoms or clusters of atoms in aperiodic arrangements on aspecimen.

The TEM may be adapted to operate in an incoherent illumination mode. Inthis mode, the coherence of illumination of the TEM is eithersubstantially mitigated or eliminated completely. Incoherence means thatdifferent sets of electrons impinging on the specimen are incoherent inrelation to one another. In one embodiment, the TEM is implemented witha substantially incoherent electron source. For example, the electronbeam source may be adapted to generate an electron beam having an energyspread of less than about 1 eV. Alternatively or in addition, the TEMmay produce electron beams that are incoherent in relation to oneanother at different times. For example, the TEM may differently shiftor scan the electron beam over time. In yet another example, the TEMspreads the energy of the electrons in the beam over differentpredefined ranges of energies over time.

By using incoherent illumination, the contrast between single heavyatoms or clusters of atoms and a relatively light atom substrate of thespecimen can be improved while, simultaneously, more current canpotentially be directed onto the sample. Incoherent sources can oftenachieve higher current in exchange for coherence. The contrastimprovement arises from the fact that the contrast due to the heavy atomdoes not depend on interference of a coherent electron wave whereas thedetails of speckle contrast from the specimen do. Each electron wavethat contributes to the image will therefore add intensity at the heavyatom position but average out intensity in the speckle contrast. Thisimprovement may be particularly suited for a system that needs higherdata throughput and less expensive electron sources. In other words, theincoherent illumination mode may enable higher throughput, lessexpensive sources, and better contrast.

The incoherence provides a contrast mechanism that allows directinterpretability of the resulting images. Under incoherent illuminationconditions, phase contrast is reduced whereas amplitude contrast isincreased by the mechanism of superposition: the randomness of imagefeatures in phase contrast signals interferes destructively whereas thescattering from point-like objects interferes constructively. In theaggregate, the scattering information from the point-like objects isretained while the phase contrast information from the amorphoussubstrate is intentionally washed out. Incoherent illumination may alsoincrease microscope throughput, at least in part due to an increasedelectron dose from incoherent illumination as compared with coherentillumination.

TEM imaging may be adapted to operate in a “bright field” mode in whicha central beam (referred to as a “zero beam”) of electrons in theelectron beam of the microscope is transmitted through the TEM column.Alternatively, TEM imaging may be adapted to operate in a “dark field”mode in which the central beam is blocked. Indeed, the dark-field modemay be implemented as a primary or dedicated image mode for the TEM. Thedark-field mode can produce an image with monotonic contrast change withincreasing atomic number, which allows direct interpretability of theimage to determine relative atomic weights. For example, dark-fieldimaging can be used to obtain chemically sensitive projections of singleatoms, clusters of atoms, or nanostructures. However, the dark-fieldmode may decrease the data throughput of imaging due to reduced electrondose, which taken alone may be undesirable. Thus, dark-field imagingtechniques based on coherent illumination and suffering from sphericalor other aberration may be undesirably slow compared to incoherentillumination.

In order to improve speed, TEM imaging may be adapted to correct foraberrations. Aberrations can be detected and a computer can be used toanalyze the aberrations and apply compensating signals toaberration-producing lens elements. The aberration correction canprovide increased throughput of imaging. Such increased throughput maybe especially advantageous in using TEM for DNA sequencing. The highthroughput may allow the microscope to be used in sequencing a fullhuman genome substantially quickly. For example, the microscope may beadapted to sequence a full human genome in from about 200 hours to about0.01 minutes, such as about 20 hours. In an especially high-throughputversion, the microscope may be used in sequencing a full human genome infrom about 10 hours to about 1 minute. The term “DNA,” as used herein,includes for convenience a wide range of relevant specimens, includingoligonucleotides and polynucleotides, and to DNA or RNA of genomic,recombinant, or synthetic origin, which may be single- ordouble-stranded, and represent the sense or antisense strands, or to anyDNA-like or RNA-like material or other polymers, such as proteins,natural, recombinant, or synthetic in origin, and which may contain anynucleic acid, including variants such as 5-Metyl Cytosine and otherepigenetically modified bases, artificially modified bases, andindividual amino acids, both natural and artificial.

In one version, aberration correction is implemented in a TEM that isadapted to operate in the dark-field mode. As described in copendingU.S. patent application Ser. No. 13/024,961 to Own et al., titled“Aberration-Correcting Dark-Field Electron Microscopy” and herebyincorporated by reference in its entirety, a combination of aberrationcorrection and dark-field mode may be especially advantageous when theaberration correction is implemented wholly or in part using“charge-on-axis” elements. “Charge-on-axis” refers to one or moreelements placed approximately at the zero beam of the microscope. In abright-field mode, in contrast, the zero beam would not be blocked byany such elements.

Reference will now be made in detail to exemplary embodiments of TEMs,which are illustrated in the accompanying drawings. Wherever possible,the same reference numbers will be used throughout the drawings to referto the same or similar parts.

In an exemplary embodiment of a TEM, an electron gun together withcondenser lenses and a pre-field of an objective lens form a patch ofelectron illumination on a specimen. The atoms of the specimen scatterthe incident electrons, with the heavier atoms scattering the electronsto higher angles. The post-sample field of the objective lens creates adiffraction pattern in a back-focal plane of the objective lens.

In one version of an approximately cylindrically symmetric system, theTEM may have an annular aperture. In this case, an aperture containing acentral beam stop may be mounted in or near the back-focal plane (or aplane conjugate to it). The central beam stop may have the shape of acircular disc. The beam stop limits the scattered electrons to anangular range denoted here as φ_(d), which defines an annulus between aninner angle φ₁ and outer angle φ₂. These angles may be, for example, inthe case of imaging clusters of atoms, from about 0.1 mrad to about 10mrad for φ₁, and from about 1 mrad to about 20 mrad for φ₂. In the caseof imaging single atoms, these angles may be, for example, from about 5mrad to about 20 mrad for φ₁, and at least about 20 mrad for φ₂. Anexample of a suitable range for φ₂ is from about 20 mrad to about 50mrad. Thus, an example of a suitable range for φ_(d) for imaging singleatoms may be from about 15 mrad to about 50 mrad. Electrons passingthrough this annular aperture are ultimately collected on a detector,generating an image of the specimen. In other words, the rays passingthrough the annular aperture are ultimately the source of theinformation gleaned from the specimen. At higher angles less intensitymay be received at the detector, since the image intensity may drop offapproximately with r², where r is the distance in image plane 100 fromoptic axis 130.

Alternatively to a cylindrically symmetric system, the central beam stopmay have a non-circular shape. For example, the central beam stop mayhave the shape of a square, hexagon, or another polygon. The inner edgeof the aperture may also have a non-circular shape. Thus, the annularopening may have non-circular edges. Such a non-circular annular openingmay result in less contrast than a circular annular opening.

An exemplary embodiment of the structural configuration of a TEM isdescribed below. This example of the TEM has an electron optical columnthat includes an electron source, condenser lenses, a specimen holder,an objective lens, and a detector. In certain embodiments of the TEM,the electron source may be a thermionic source, such as a tungsten (W)or lanthanum hexaboride (LaB₆) source. These sources may provide asubstantially large current, which may be advantageous in allowingshorter exposures for each image and hence higher throughput. Theseexamples of electron sources may also provide substantially incoherentillumination, as explained in more detail below.

The electron source may be followed by condenser lenses to form a beamthat will be incident on the sample. The condenser lenses may consistof, for example, two, three, or four lenses. The condenser lenses may bemagnetic or electrostatic. The electrons scattered from the specimen arethen imaged through an optical system.

The electromagnetic lenses can also include additional correctingelements. Furthermore, there is an opportunity for standard magnifyinglenses to be included in the TEM. These magnifying lenses are followedby an electron detector. The electron detector may have one of manyforms that are known to one of ordinary skill in the art.

In a dark-field mode, the optical system may accomplish at least twoadditional functions. First, the optical system may block out thecentral scattered beam to implement the ADF mode. Second, the opticalsystem may correct aberrations. The combination of these two featurescan be particularly advantageous. As part of the aberration-correctingsystem, a charge-inducing component may be positioned at least partiallyon the optic axis of the ADF-TEM column (or a more conventionalmultipole-based aberration corrector), either before, in, or after theobjective lens in relation to the beam path.

The TEM may also include a system to correct for parasitic aberrations,in contrast to spherical aberrations, whether parasitic aberrations arecylindrically symmetric aberrations or not. Parasitic aberrations may becaused, for example, by the optical elements having been machined insuch a way as to be very slightly off-axis or very slightly non-round.

In one example, a standard aberration corrector for a TEM may include aNion Co. quadrupole-octupole corrector or CEOS Co. sextupole orquadrupole-octupole corrector. An annular aperture may be providedeither in the incoming illumination of the sample (such as for a STEMmode of an ADF-TEM) or in the outgoing scattered beam (such as for adark-field TEM mode).

The aberration-correcting TEM may additionally include a mechanism fordiagnosing the aberrations. Conventional approaches for diagnosingaberrations typically assume that a bright-field image is available. Onenovel method for TEM is to acquire images as a function of illuminationtilt and defocus, and to extract the blurring effect of the tilt anddefocus. The blurring gives a value for the defocus and astigmatism at avariety of angles. This process can provide sufficient data tonumerically compute an aberration function for the imaging system. Asample used for these purposes may contain single atoms or clusters ofatoms, or may be another kind of sample made for the purpose ofdiagnosing aberrations. For example, the sample may be the specimen thatis ultimately the subject of interest for study. Alternatively, thesample may be a sample used solely for calibration of theaberration-correcting TEM.

A particularly useful application of the TEM is to analyze a DNA samplein order to determine the sequence of its base pairs. In one version, asingle strand of DNA is stretched using techniques that have beendescribed in PCT Publication No. WO 2009/046445 dated Sep. 4, 2009,entitled “Sequencing Nucleic Acid Polymers with Electron Microscopy,”and filed as International Application No. PCT/US2008/078986 on Jun. 10,2008 (this PCT publication is hereby incorporated by reference in itsentirety). A particular set of bases has been labeled with a label thatcontains at least one heavy scatterer, such as a single heavy atom or acluster of atoms. Examples of such labels include osmium, triosmium, andplatinum.

FIG. 1 is a schematic diagram of an exemplary embodiment of anaberration-correcting ADF-TEM column 10. Column 10 has an electronsource 20, one or more condenser lenses 30, specimen 40, objective lens50, annular aperture 60, one or more projecting lenses 70, and detector80. An aberration corrector may be incorporated in objective lens 50.Image plane 100 is shown in the figure. Electron source 20 and condenserlenses 30 are configured to provide a variety of illuminationconditions. For example, electron source 20 and/or condenser lenses 30may be adapted to provide a high-current, incoherent illumination mode.

TEM column 10 is connected to a power source that provides power tocomponents of column 10, such as electron source 20, lenses 30, 50, and70, and detector 80, as well as a stage that holds and moves specimen40. Column 10 may have a total power consumption of less than about 800W. In a low-power embodiment, such as where column 10 is miniaturized,column 10 may even have a power consumption of less than about 300 W,such as from about 10 W to about 100 W. Electron source 20 may beadapted to generate an electron beam with a current of less than about100 mA. In an especially low-current version, electron source 20 mayeven be adapted to generate an electron beam with a current of less thanabout 10 μA, such as less than about 10 pA.

The features described herein for the aberration-correcting dark-fieldTEM may be implemented in many different types of microscopes utilizingcharged ions or other particle beams. Moreover, theaberration-correcting dark-field TEM may be used in any suitablefacility in any desired arrangement, such as networked, direct, orindirect communication arrangements.

Furthermore, the TEM system may include any quantity of components, suchas microscopes, machine managers, computer systems, networks, and imagestores, that may be in communication with or coupled to each other inany suitable fashion, such as wired or wireless, over a network such asWAN, LAN, or Internet, directly or indirectly coupled, local or remotefrom each other, via any communications medium, and utilizing anysuitable communication protocol or standard.

The embodiments of TEM described herein may be implemented with eitherelectrostatic or magnetic components. For example, for a commercialsetting, a relatively small electrostatic version of the TEM may beconstructed. The TEM system may include any quantity of electrostatic ormagnetic components, such as electron or other particle gun, lenses,dispersion device, stigmator coils, electron detectors, and stages,arranged within or external to the TEM in any suitable fashion. Imagestores, files, and folders used by the TEM system may be of any quantityand may be implemented by any storage devices, such as memory, database,or data structures.

The TEM may include a controller. The controller may include any one ormore microprocessors, controllers, processing systems and/or circuitry,such as any combination of hardware and/or software modules. Forexample, the controller may be implemented in any quantity of personalcomputers, such as IBM-compatible, Apple, Macintosh, Android, or othercomputer platforms. The controller may also include any commerciallyavailable operating system software, such as Windows, OS/2, Unix, orLinux, or any commercially available and/or custom software such ascommunications software or microscope monitoring software. Furthermore,the controller may include any types of input devices such as atouchpad, keyboard, mouse, microphone, or voice recognition.

The controller software, such as a monitoring module, may be stored on acomputer-readable medium such as a magnetic, optical, magneto-optic, orflash medium, floppy diskettes, CD-ROM, DVD, or other memory devices,for use on stand-alone systems or systems connected by a network orother communications medium, and/or may be downloaded, such as in theform of carrier waves, or packets, to systems via a network or othercommunications medium.

The controller may control operation of the TEM column. Alternatively orin addition, the controller may receive an image from the detector ofthe TEM to be processed computationally. For example, the controller mayprocess collected particle data and/or process any desired images. Thecontroller may include an image formation unit for this purpose. Theimage formation unit may be disposed within or external of the TEMcolumn and communicate with the microscope column in any fashion such asdirectly or indirectly coupled, or communicate via a network.

Moreover, the various functions of the TEM may be distributed in anymanner among any quantity such as one or more of hardware and/orsoftware modules or units, computer or processing systems or circuitry,where the computer or processing systems may be disposed locally orremotely of each other and communicate via any suitable communicationsmedium such as LAN, WAN, Intranet, Internet, hardwire, modem connection,or wireless. The software and/or algorithms described above may bemodified in any manner that accomplishes the functions described herein.

The TEM may use any number of images of a sample to determine optimalbeam parameter settings and/or image quality values. The images maycover any desired variation range for a particular parameter. The samplemay be of any quantity, may be of any shape or size, and may include anydesired features. For example, the sample may include a specificconfiguration for a desired application or parameter setting. The samplemay be disposed at any desired location on or off the stage to acquireimages. In one example, the sample is in the form of a product specimensuch as a semiconductor device. Alternatively, the sample may be a testspecimen such as gold nano-particles on a carbon film.

The TEM may also use any number of images for the image qualitycomparison, where the image quality values for current and prior imagesmay be combined in any suitable fashion, such as averaged, weighted, orsummed. The user threshold may be set to any suitable values dependingupon the desired image quality. The comparison of image quality valuesmay utilize any mathematical or statistical operations to determineimage quality compliance such as a comparison, statistical variance, ordeviation.

The TEM may analyze any suitable characteristics, such as intensity,pixel counts, or power, each of which may be analyzed in real space orin frequency space (so that intensity may be within or without a spatialfrequency range) and utilize any differentiating characteristic betweensettings in any desired region. The region of separation may be of anyshape or size and be located within any desired range. Theaberration-correcting dark-field TEM may also utilize any suitablemodeling or approximation techniques to determine best fit lines and/orcurves such as linear or non-linear regression, curve fitting, leastsquares, or integration. The models may approximate the data within anysuitable tolerances. The TEM may identify any quantity of separationregions and utilize any suitable techniques to combine and/or selectresulting slope values such as lowest slope, average, weighting, or sum.

The parameter determination may be triggered in any suitable fashion.For example, the machine manager may monitor the microscope to initiatethe determination, the computer system or controller may periodicallyretrieve images based on a periodic acquisition of sample images or pollthe image store to determine the presence of sample images, or manuallytrigger determination. The quality inspection and/or parameterdetermination may be initiated in response to any suitable conditions(e.g., within any desired time interval such as within any quantity ofhours or minutes, subsequent any quantity of images generated by themicroscope such as every Nth scan performed by the microscope,subsequent any quantity of quality inspections.

The TEM imaging technique may be performed automatically, whereparameters are determined and applied to the microscope. Alternatively,any part of the technique, such as scanning of images, determination ofparameters, or application of the parameters, may be performed manually.For example, the computer system may provide the optimal settings to atechnician that manually applies the settings to the microscope. Themicroscope controller may perform any desired processing, such asmonitoring and parameter adjustment or image formation and processing.

Implementation of aspects of the TEM, such as the image processing oraberration correction, may be distributed among the computer system,microscope controller, or other processing device in any desired manner,where these devices may be local or remote in relation to one another.The computer system and microcontroller communicate with and/or controlthe microscope to perform any desired functions, such as scan thespecimen and generate the images or transfer images to memory.

While the incoherent TEM can be built de novo from specifically designedcomponents, there may be practical advantages to modifying conventionalEM systems to provide the advantageous characteristics of the inventivesystems. For example, such modification may allow existing EM facilitiesto upgrade their current equipment to obtain the advantages of theincoherent TEM at a desirably low cost without requiring theconstruction of an entirely new EM system. The modification may includeretrofit of new components and realignment or repositioning of existingcomponents, such as of components in the upper column of the EM.

As described herein, incoherent illumination may be generated by, forexample, the use of a substantially incoherent electron source orshifting, scanning, or altering the energy of the electron beam.

FIG. 2 illustrates an exemplary embodiment of an implementation of anincoherent illumination mode using an incoherent electron source 20A.The electron beam consists of a filled cone of many different sub-beamsthat are incoherent in relation to one another, referred to asincoherently related pencils of illumination, and are emitted byelectron source 20A. Each of incoherently related pencils 440 is aconstituent that is incoherent in relation to the other constituents. Asummation of phase contrast that cancels and amplitude signal thatreinforces occurs simultaneously at image plane 100, so a usable imagecan be produced at once. Incoherent electron source 20A may be, forexample, an electron source having a tungsten or lanthanum hexaboridefilament. Incoherent electron source 20A may be combined with two ormore condenser lenses 450, which can be excited in differentconfigurations to allow alignment in a coherent illumination mode andthen imaging operation in incoherent illumination mode.

There is an embodiment in which incoherence or the electron beam isenhanced by low demagnification of electron source 20A. The EM columnincludes condenser lenses 106 a-c to demagnify incoming electron beam104 from electron source 20A. Demagnification may be necessary in orderto sufficiently concentrate the current of electron beam 104 on adesirably small region of the specimen. Incoming electron beam 104 mayinclude a range of angles from electron source 20A or a surface area ofelectron source 20A.

FIGS. 3 a and 3 b illustrate electron beam trajectories with greater andlesser levels, respectively, of demagnification. In FIG. 3 a, condenserlens 106 a demagnifies incoming electron beam 104 into an initial beamportion 108 a. As shown in the figure, however, only a sub-portion 108 bof beam portion 108 a is received by next condenser lens 106 b. The partof beam portion 108 a that lies outside of sub-portion 108 b iseffectively discarded from the transmitted electron beam. Similarly,condenser lens 106 b demagnifies beam portion 108 b into a beam portion110 a. Of beam portion 110 a, only a sub-portion 110 b is received bynext lens 106 c. Thus, beam portion 110 b represents a subset of therange of angles from electron source 20A or surface area of electronsource 20A that is contained in initial electron beam 104.

In FIG. 3 b, on the other hand, condenser lenses 106 a-c do notdemagnify incoming electron beam 104 as strongly as in FIG. 3 a. Asshown in the figure, after condenser lens 106 b, part of beam portion112 a is effectively discarded such that only a sub-portion 112 b isreceived by next lens 106 c. Thus, beam portion 112 b represents asubset of the range of angles from electron source 20A or surface areaof electron source 20A that is contained in initial electron beam 104.Since incoming electron beam 104 is less strongly demagnified in FIG. 3b than in FIG. 3 a, beam portion 112 b in FIG. 3 b represents a largersubset of the range of angles from electron source 20A or surface areaof electron source 20A that is contained in initial electron beam 104than does beam portion 110 b in FIG. 3 a. Accordingly, beam portion 112b in FIG. 3 b contains a larger number of incoherently related pencilsof illumination.

Alternatively or in addition, the incoherent illumination mode may beimplemented in the TEM without an incoherent electron source. Forexample, the incoherent illumination mode may be implemented bygenerating electron beams that are incoherent in relation to one anotherat different times. This may be preferable where a conventional EMsystem designed for coherent-mode operation is being modified into oneof the incoherent-mode embodiments described herein.

During exposure of the image, either the angle, the position, or theenergy of the source may be altered. These changes may be made on thetime scale of the exposure. In an illustrative example, if the image isa one-second exposure, any combination of the energy, position, andangle can be oscillated, such as, for example, on the order of about 10times the exposure time. If the energy is scanned, chromatic aberrationcan be compensated for by simultaneously scanning the focus of the beam.With an incoherent illumination mode, a much higher beam current cantypically be achieved on the specimen than with coherent illumination,and therefore imaging may take place much faster. More typically, theexposure time would be, rather than one second, on the order ofmilliseconds or microseconds. An advantage of incoherent illuminationmode, particularly for the application of DNA sequencing (or anyapplication involving identifying single heavy atoms or clusters ofatoms on an otherwise low density background), may be an increase in thespeed of image acquisition that goes with the increased amount ofcurrent that impinges on the specimen.

Either rocking or scanning the electron beam may work as long as therocking or scanning is sufficiently fast. Furthermore, it may suffice toscan or rock the beam within the angular range that is collected by theaperture of the objective lens. This angular range may be considered alimiting factor since, beyond this angular range, the current impingeson the aperture and does not get detected. However, it may be desirableto scan or rock the beam slightly beyond the angular range that iscollected by the aperture of the objective lens, since scattering intothe aperture may nevertheless occur. Nevertheless, rocking or scanningthe beam across an angular range of more than twice the acceptance angleof the objective lens may not be desirable since it would waste current.

A conventional EM system designed to operate in a coherent illuminationmode may be modified into one of the embodiments described herein. ThisEM system may include an optimized system of condenser lenses, such astwo to five condenser lenses. The electron source of the conventional EMsystem may be replaced with an incoherent electron source, such as oneof the incoherent electron sources described herein. In addition,deflectors or other mechanisms may be added for laterally shifting orangularly wobbling the beam, and/or a mechanism may be added to vary theaccelerating voltage applied to the beam. Aberration-correctingillumination systems are available from JEOL, FEI, and Zeiss (such asZeiss's Kohler illumination system). The trajectories of the electronrays through one or more of the lenses of the illumination system may bealtered.

The acceptance angles of the objective lens may be determined based onthe desired resolution of the microscope. For example, if 1 Ångströmresolution at 100 kilovolts is desired, one may need approximately 20milliradians acceptance half-angle, and therefore one might preferablynot go beyond 40 milliradians of illumination half-angle. With a greaterangular range, current may undesirably be wasted. In one example,single-atom resolution—namely resolution at least as good as about 0.3nanometers and in some instances at least as good as about 0.15nanometers—may be desirable for a DNA-sequencing application. Once asuitable accelerating voltage is chosen, that resolution requirement maydetermine the acceptance angle of the objective lens.

A conventional EM system whose illumination trajectories have beenmodified to achieve relatively incoherent illumination can be furtherimproved to take advantage of the higher current enabled by incoherentillumination by increasing the speed of the detector or stage. Forexample, a piezoelectric stage may be used. The piezoelectric stage maybe able to move very quickly and settle very quickly and stably so thatshort exposures of the order of milliseconds or microseconds can bepractically achieved. The piezoelectric stage may also be adapted tomove the stage with very high positional precision. Furthermore, thethroughput of data that emerges from the detector, which in this casemay be a high-speed camera, may be quite large, such that electronicscapable of dealing with this data throughput downstream of the cameramay be desirable.

Some of the methods of achieving an incoherent illumination mode includetaking a number of image constituents that are individually coherent andcombining these image constituents incoherently. There are severaldifferent ways of generating and incoherently combining these imageconstituents, as described in more detail below.

FIG. 4 illustrates an exemplary embodiment of a reference version of aTEM that other exemplary embodiments described below will be compared tofor the sake of illustration. Illumination 460 is provided parallel tooptic axis 130 and onto specimen 40. Beams scattered from specimen 40are collected by the objective lens in objective lens plane 470, whichfocuses the beams onto image plane 100. In back focal plane 480 of theobjective lens, a diffraction pattern is formed that is the Fouriertransform of the exit waveform of specimen 40, representing angles ofscatter from specimen 40. Three scattered beams 490 are showndemonstrating that rays scattered to the same angle by different pointson specimen 40 converge to specific points in back focal plane 480representative of their scattering angle. In projection, the threepoints correspond to scattering vectors g, 0 (forward-scattered), and−g. The “forward-scattered beam” refers to the zero beam (i.e., the 0scattering vector) and a small range of angles around the zero beam.

FIG. 5 illustrates an exemplary embodiment of a dark-field version ofthe TEM illustrated in FIG. 4. In this version, back focal plane 480contains annular aperture 60, which includes a beam stop 600, a centraldisc that leaves an opening 610 with outer diameter D for implementingthe annular-dark-field mode. While the left and right scattered beamspass through annular aperture 60, the forward-scattered beam (0) isblocked by beam stop 500 in or near back focal plane 480. Only the rayspassing through annular aperture 60 propagate to image plane 100,forming a dark-field image.

There is a relationship between the aperture diameter D and resolution.Aperture 60 selects the range of angles that are used to form the imageor probe in TEM or STEM, respectively. In the case of TEM, specimen 40is illuminated and electrons are scattered from different points onspecimen 40. The electrons scattered to a particular angle fromdifferent parts of specimen 40 are brought to a common point in backfocal plane 480, and then propagated further until they form an image.Aperture 60 thus selects the angles which form the image by limiting therays passing through back focal plane 480. In the case of STEM, where asource produces a plane wave that enters the objective lens, theobjective aperture placed in or near back focal plane 480 limits thesize of illumination entering the objective lens. The rays that theobjective lens focuses to a point on the specimen are thus limited inangle by the aperture.

FIG. 6 illustrates a schematic diagram of an exemplary embodiment of abright-field implementation in which an electron beam 620 is tilted atan angle in relation to optic axis 130 of the EM column. Electron beam620 may be scanned radially such that electron beam 620 keepssubstantially the same angle in relation to optic axis 130, forming asubstantially cylindrically symmetric cone of electron illuminationabout optic axis 130. Alternatively, the angle of tilted electron beam620 may be flipped between two angles that are symmetric (or “minor”angles) with respect to optic axis 130, the figure showing an example ofone of the two mirror angles. For example, electron beam 620 may bepassed through a tilting prism that is used to alternately flip electronbeam 620 between these two minor angles. Specimen 40 scatters theincident electrons, resulting in scattered beams 630, shown in thefigure as two beams on the sides and the one beam in the middle.Scattered beams 630 are focused by the objective lens to the imageplane. Scattered beams 630 create a diffraction pattern in back focalplane 480 and are filtered by aperture 633 with a circular disc opening637. Image constituents from different scan positions of electron beam620 are summed. These image constituents are incoherent in relation toone another. By illuminating specimen 40 through a cone of illumination,or alternately illuminating specimen 40 at mirror angles, and collectingthese image contributions on image plane 100 over time, anincoherently-summed image can be produced. The tilt angle may be lessthan about 100 milliradians in relation to optic axis 130 and may exceedthe aperture radius D/2.

FIG. 7 illustrates an exemplary embodiment of a variation of the TEMillustrated in FIG. 6 with an annular aperture. In this variation, backfocal plane 480 contains annular aperture 60, which includes a beam stop600, a central disc that leaves an opening 610 with outer diameter D.While the forward-scattered beam (0) passes through annular aperture 60,the left and right scattered beams are blocked by beam stop 600 in ornear back focal plane 480. Only the rays passing through annularaperture 60 propagate to image plane 100, forming a dark-field image.

FIG. 8 illustrates an exemplary embodiment of a dark-field variation ofthe tilted-beam version of FIG. 6. As shown in the figure, aperture 633may be adapted, and the tilt angle may be selected, to be sufficientlyhigh to cause the zero beam to impinge on aperture 633 and not passthrough opening 637 in or near back focal plane 480. While the rightscattered beam pass through aperture 633, the forward-scattered beam andleft scattered beam are blocked by aperture 633 in or near back focalplane 480. Only the rays passing through opening 637 propagate to imageplane 100, forming a dark-field image. Alternatively, an annularaperture, such as shown in FIGS. 5 and 7, may be used in place ofaperture 633 with circular disc opening 637.

The angle of tilt of electron beam 620 may be set to select forscattering angles. These scattering angles, in turn, correspond todifferent chemical compositions. For example, heavier atoms tend toscatter to a higher angle while lighter atoms scatter to a lower angle.In an exemplary embodiment, the background is carbon, for whichrelatively low-angle scattering is blocked by aperture 633.

In one version, the aperture is adapted and the tile angle is selectedto make the forward-scattered beam impinge mostly on aperture 633, justoutside of opening 637, such that the zero beam is blocked by the edgeof aperture 633 but neighboring angles pass through opening 637. In thiscase, a portion of the forward-scattered beam is passed while most isblocked.

FIG. 9 illustrates a generalized version of the manner in whichamplitude contrast is enhanced in incoherent annular-dark-field imagingwhile phase contrast is decreased to improve contrast of point-likeobjects in the specimen. This figure shows an ideal amplitude signal 640from a specimen containing a point-like object on an amorphousbackground. For the sake of illustration, different incoherently relatedimage constituents, labeled as 650, 660, 670, 680, etc., that arecreated from the object are shown as vertically arranged. For each ofthese image constituents, the horizontal axis represents position andthe vertical axis represents signal amplitude. Each of the imageconstituents contains an amplitude contrast component and also a phasecontrast component. The latter component dominates in TEM images, as canbe seen in image constituents 650, 660, 670, 680, etc. The methodsdescribed herein extract amplitude signal 640 from the unwanted phasecontrast.

For example, a number of image constituents 650, 660, 670, 680, etc.,are taken either in sequence or simultaneously in a particular imagingmode. In each of image constituents 650, 660, 670, 680, etc., theamplitude signal may be small in comparison to the speckle noise fromthe phase contrast. However, the speckle noise varies substantiallybetween different image constituents 650, 660, 670, 680, etc. Meanwhile,hidden within this noise is a weak yet consistent amplitude signal 640between different image constituents 650, 660, 670, 680, etc. Thus, whenimage constituents 650, 660, 670, 680, etc., are superimposed, the phasecontrast tends to cancel while amplitude signal 640 constructivelyinterferes, forming an increasingly discernible signal 690 as moreincoherently-related constituent images are included.

FIGS. 10 a and 10 b are images and histograms of two examples, for thesake of illustration, of amplitude signals 690 along a preselecteddirection. The images at the bottom of the figures are grayscale imagesof intensity received by the detector of the transmission electronmicroscope. The histograms at the top of the figures, corresponding tothe images below them, respectively, show the same intensity informationin a two-dimensional graph. As shown in these examples, an amplitudesignal emerges from the noise in the centers of the images andhistograms.

FIG. 11 illustrates an example of yet another embodiment of theimplementation of incoherent superposition in a bright-field mode. Inthis figure, different energies are used such that the scattering angleschange slightly. Since the objective lens focuses higher energyelectrons less strongly than lower energy electrons, the scatteringangles from the specimen vary accordingly, as indicated in the figure byscattered beams 490 and 700, representing, respectively, lower andhigher energy beams. In this figure, in contrast to FIG. 4, part of thebeam that would have been imaged as a point in image plane 100 is nowimaged onto a diffuse region 710 and another part of beam 460 that wouldhave been imaged onto a point is now imaged onto a narrower diffuseregion 720. While regions 710 and 720 represent diffuse images of thesame point of specimen 40, their centers are still approximately in thesame location when projected onto image plane 100. A point-likeamplitude object, when imaged in this way, will consistently be imagedto the same point. Simultaneously, speckle contrast from the backgroundwill be averaged out.

Adjustment of electron energy can be achieved by various alternativemethods including, for example, choosing a source with a large energyspread in the electron source, increasing the chromatic aberration inthe illumination system, and modulating the voltage applied to theelectron source with time at a frequency greater than the exposurefrequency.

FIG. 12 illustrates an exemplary embodiment of a variation of thespread-energy version of FIG. 11 that is implemented to includechromatic aberration correction. As shown in FIG. 12, achromatic-aberration corrector 615 is included to correct for chromaticaberrations. Chromatic-aberration corrector 615 may be disposed, forexample, below back focal plane 480.

FIG. 13 illustrates an exemplary embodiment of a dark-field variation ofthe spread-energy version of FIG. 11.

FIG. 14 illustrates an example of yet another embodiment of theimplementation of incoherent superposition. In this embodiment, imagesexposed at different times are summed. Again, through the summation,more amplitude contrast is generated while the phase contrast isdecreased. Although FIG. 14 illustrates a bright-field version, thisconcept may also be implemented in a dark-field mode (not shown).

FIG. 15 illustrates still another exemplary embodiment of theimplementation of incoherent superposition. In this figure, the electronbeam is shifted laterally to different lateral positions, such asposition 725 and position 730, at different times to obtain differentsets of scattered beams 490 and 495 and the resulting differentrelatively incoherent constituents. This shift can be achieved in oneexample by using dipole deflectors to shift the beam before it reachesthe sample.

FIG. 16 illustrates an exemplary embodiment of a dark-field variation ofthe time-shifted version of FIG. 15.

FIG. 17 illustrates yet another bright-field embodiment that isconceptually similar to FIG. 15, but in which a prism 740 is used in thebeam path to shift all or a portion of the beam, such as from position725 to position 730.

The examples shown in FIGS. 6, 7, 8, 11, 12, 13, 14, 15, 16, and 17illustrate various embodiments in which constituent images are exposedserially or in parallel to improve amplitude contrast relative to phasecontrast, thereby improving interpretability of the image.

The electron energy used in the TEM may be determined at least in partbased on the transmission properties of the specimen. The specimen mayhave a thickness on the order of 2 nanometers, such as for example athickness of about 1 nanometer. In one example, the specimen is made ofcarbon, although single-atom-thick graphene may also be used. As aresult, 1 keV electrons are likely to be the lowest energy appropriatewhen considering voltage alone.

Unfortunately, since electron wavelength varies inversely with energy,the diffraction limit may require the angles to be corrected to belarge. Such an aberration-correcting dark-field TEM may becomechallenging to manufacture. It may be desirable for the dark-field TEMto be operated at a much higher voltage, such as from about 1 kV toabout 300 kV. For example, the dark-field TEM may be operated at about30 kV. This voltage range is in the realm of conventional microscopy,and the implementation of electrostatic correction elements may beunfeasible in this range due to the risk of damage from high localfields, high voltage discharge, and transmission of high-energyelectrons.

For miniaturized embodiments of the aberration-correcting dark-field TEMcolumn, the voltage may be based in part on the dimensions of theminiaturized embodiments. The miniaturization of the column could goeven further than described above. Such a miniaturizedaberration-correcting dark-field TEM column remains a column withaberration correction by charge-on-axis elements with substantially thesame features described herein. A specialized detector may also beuseful so that operation of the instrument is in STEM mode or SEM mode,rather than in TEM mode. In that case, a fabricated solid-statebackscatter detector may be provided.

The TEM may preferably use a beam current of from about 10 picoamps toabout 1 milliamp. When an incoherent illumination mode is intended, thehigh spatial charge density may desirably increase the incoherence.Thus, for an incoherent illumination mode, a beam current above 100microamps may be used advantageously.

Furthermore, it may be desirable for the beam to be sufficientlymonochromatic, in other words to have a sufficiently narrow range ofenergies, to avoid focus problems. A spread in energies of the electronsin the beam typically causes a corresponding change of focus of theimage. Thus, the image may be thought of as a sum of many images thathave changing foci. If that range is too large, then the intensity of asingle atom in the image may get blurred out over a large region andthereby become indistinguishable from the background. Thus, it may bepreferable to have an energy spread of less than about 10 eV to avoidsuch blurring. Where a tighter focus is desired, however, it may bepreferable to have an energy spread of less than about 1 eV. Forexample, the electron beam may even have an energy spread of less thanabout 200 meV. This may be desirable where there is no chromaticaberration correction in the optical system. In other circumstances,however, such as if chromatic-aberration correction is implemented inthe optical system, much larger energy spreads may be used. For example,the chromatic-aberration correctors described herein may be able tohandle hundreds of electron volts of energy spread.

Although the foregoing embodiments have been described in detail by wayof illustration and example for purposes of clarity of understanding, itwill be readily apparent to those of ordinary skill in the art in lightof the description herein that certain changes and modifications may bemade thereto without departing from the spirit or scope of the appendedclaims. It is also to be understood that the terminology used herein isfor the purpose of describing particular aspects only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only,” and the like in connection with therecitation of claim elements, or use of a “negative” limitation. As willbe apparent to those of ordinary skill in the art upon reading thisdisclosure, each of the individual aspects described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalaspects without departing from the scope or spirit of the disclosure.Any recited method can be carried out in the order of events recited orin any other order which is logically possible. Accordingly, thepreceding merely provides illustrative examples. It will be appreciatedthat those of ordinary skill in the art will be able to devise variousarrangements which, although not explicitly described or shown herein,embody the principles of the disclosure and are included within itsspirit and scope.

Furthermore, all examples and conditional language recited herein areprincipally intended to aid the reader in understanding the principlesof the invention and the concepts contributed by the inventors tofurthering the art, and are to be construed without limitation to suchspecifically recited examples and conditions. Moreover, all statementsherein reciting principles and aspects of the invention, as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryconfigurations shown and described herein. Rather, the scope and spiritof present invention is embodied by the claims.

In this specification, various preferred embodiments have been describedwith reference to the accompanying drawings. It will be evident,however, that various other modifications and changes may be madethereto and additional embodiments may be implemented, without departingfrom the broader scope of the claims that follow. The specification anddrawings are accordingly to be regarded in an illustrative rather thanrestrictive sense.

We claim:
 1. A transmission electron microscope comprising: an electronbeam source to generate an electron beam; beam optics to converge theelectron beam; a specimen holder to hold a specimen in the path of theelectron beam; and a detector to detect the electron beam transmittedthrough the specimen, wherein the transmission electron microscope isadapted to generate two or more images that are substantiallyincoherently related to one another, store the images, and combineamplitude signals at corresponding pixels of the respective images toimprove a signal-to-noise ratio.
 2. The transmission electron microscopeof claim 1, wherein the transmission electron microscope is adapted toconsume less than about 800 W of power.
 3. A method of generating animage of a specimen, the method comprising: generating an electron beam;converging the electron beam; holding a specimen in the path of theelectron beam; detecting the electron beam transmitted through thespecimen to generate two or more images that are substantiallyincoherently related to one another; storing the two or more images; andcombining amplitude signals at corresponding pixels of the respectiveimages to improve a signal-to-noise ratio.
 4. The method of claim 3,wherein the method consumes less than about 800 W.
 5. A transmissionelectron microscope comprising: an electron beam source to generate anelectron beam; beam optics to converge the electron beam; a specimenholder to hold a specimen in the path of the electron beam; and adetector to detect the electron beam transmitted through the specimen,wherein the transmission electron microscope is adapted to operate thespecimen holder to move the specimen between two or more exposure timesthat correspond to a single image to improve a signal-to-noise ratio inan incoherent mode.
 6. The transmission electron microscope of claim 5,wherein the transmission electron microscope is adapted to operate thespecimen holder to move the specimen during exposure for a single imageto improve a signal-to-noise ratio.
 7. The transmission electronmicroscope of claim 5, wherein the transmission electron microscope isadapted to operate the specimen holder to move the specimen between twoor more exposure times that correspond to separate images that are to becombined to improve a signal-to-noise ratio.
 8. The transmissionelectron microscope of claim 5, wherein the transmission electronmicroscope is adapted to have a power consumption of less than about 800W.